Effects on translation pausing of alterations in protein and RNA components of the ribosome exit tunnel

Abstract

Amino acids are polymerized into peptides in the peptidyl transferase center of the ribosome. The nascent peptides then pass through the exit tunnel before they reach the extraribosomal environment. A number of nascent peptides interact with the exit tunnel and stall elongation at specific sites within their peptide chain. Several mutational changes in RNA and protein components of the ribosome have previously been shown to interfere with pausing. These changes are localized in the narrowest region of the tunnel, near a constriction formed by ribosomal proteins L4 and L22. To expand our knowledge about peptide-induced pausing, we performed a comparative study of pausing induced by two peptides, SecM and a short peptide, Crb(CmlA), that requires chloramphenicol as a coinducer of pausing. We analyzed the effects of 15 mutational changes in L4 and L22, as well as the effects of methylating nucleotide A2058 of 23S rRNA, a nucleotide previously implicated in pausing and located close to the L4-L22 constriction. Our results show that methylation of A2058 and most mutational changes in L4 and L22 have differential effects on pausing in response to Crb(CmlA) and SecM. Only one change, a 6-amino-acid insertion after amino acid 72 in L4, affects pausing in both peptides. We conclude that the two peptides interact with different regions of the exit tunnel. Our results suggest that either the two peptides use different mechanisms of pausing or they interact differently but induce similar inhibitory conformational changes in functionally important regions of the ribosome.

FIG. 1.

Cartoon model of the 50S subunit of the ribosome of E. coli, showing the PTC, the peptide exit tunnel, the tips of r-proteins L4 and L22, and the approximate binding sites of antibiotics chloramphenicol (CM) and erythromycin (Ery).

FIG. 2.

SecM-mediated pausing in L4 and L22 mutants. A. Map of the construct for quantifying efficiency of pausing. White boxed areas indicate sequences derived from the multicloning site of pGEM5. The SecM sequence is shown in black. Light gray boxes indicate sequences from the lacZ gene. For more details, see Materials and Methods. B. Quantitation of SecM/β-galactosidase fusion protein synthesis. The β-galactosidase activity was measured in strains containing the indicated L22 or L4 genes or by using a fusion protein construct containing a mutation in the SecM sequence (P166A) that inactivates the pausing peptide (21), as described in Materials and Methods. At least three independent cultures were analyzed for each mutant, and β-galactosidase assays were performed in duplicate for each culture. The standard error of the mean is indicated. M.U., Miller units.

FIG. 3.

Crb^CmlA-mediated pausing in L4 and L22 mutants. A. Map of the construct used for quantifying efficiency of pausing. White boxed areas indicate sequences derived from the multicloning site of pGEM5. This region is interrupted by the segment of DNA containing the Crb pausing peptide (black box), intergenic region, and the N terminus of cmlA (black box). Light gray boxes indicate sequences from the lacZ gene. For more details, see Materials and Methods. B. Quantitation of CmlA/β-galactosidase fusion protein synthesis. The β-galactosidase activity was measured in strains containing the indicated L22 or L4 genes, as described in Materials and Methods. The light gray bars indicate measurements from cells grown in the absence of antibiotic. The black bars indicate measurements from cells grown in the presence of chloramphenicol (0.8 μg/ml). At least three independent cultures were analyzed for each mutant, and β-galactosidase assays were performed in duplicate for each culture. The standard error of the mean is indicated for the induced values. M.U., Miller units.

FIG. 4.

Effect of methylation of A2058 on SecM- and Crb^CmlA-mediated pausing and erythromycin binding. Wild-type cells containing a plasmid with an arabinose-inducible ErmC gene were grown in the absence or presence of arabinose. Aliquots were removed for quantitation of β-galactosidase activity, and the remainder of the cultures were used for ribosome preparation and primer extension and erythromycin binding analysis (see Materials and Methods for details). A. Secondary structure of 23S rRNA in the region of A2058. The oligo used for primer extension was complementary to the highlighted bases. B. Primer extension analysis of the methylation of A2058. P, primer; 2059, extension products terminated at nucleotide 2059 (indicating methylation blockage at A2058); 2057, extension products terminated at G2057 (no methylation). C. Binding of [¹⁴C]erythromycin to ribosomes from cells grown with and without arabinose. D. Quantitation of SecM/β-galactosidase fusion protein synthesis in cells grown with and without arabinose. Standard errors of the means are indicated. E. Quantitation of CmlA/β-galactosidase fusion protein synthesis in cells grown in the presence of arabinose. Standard errors of the means are indicated. M.U., Miller units.

FIG. 5.

Structure of the E. coli ribosome exit tunnel. The figure shows a slab view through the ribosome tunnel from the PTC past the region of the tunnel lined on one side by the L22 tentacle. Nucleotides A2450 and A2451 (magenta) are used as markers for the PTC. Also shown are A2058 and A2059 (red), marking the erythromycin binding site and the entry to the beginning of the narrow part of the tunnel. Nascent peptides are synthesized at the PTC and then migrate past nucleotides A2058 and A2059 and through the constriction formed by the tips of r-proteins L4 and L22 before moving further into the tunnel lined on one side by the L22 tentacle. The tentacles of L4 (blue) and L22 (yellow) are shown with selected side chains marking positions of amino acid substitutions, insertions, and deletions analyzed in this study. Of particular interest is the Δloop2 deletion in L22, in which all residues from M82 through K98 were replaced with two glycine residues. In this mutant, much of the L22 contribution to the tunnel lining is presumably eliminated. For more details about mutants, see the text and also references and .